1. Field of the Invention
The present invention relates to the fields of molecular biology, virology and immunology of alphaviruses. More specifically, the present invention provides an attenuated, recombinant alphaviruses having mosquito infection incompetence and discloses a method of generating such alphaviruses and use of these attenuated alphaviruses in immunogenic compositions.
2. Description of the Related Art
The Alphavirus genus in the Togaviridae family contains a number of significant human and animal pathogens. These viruses are widely distributed on all continents except for the Antarctic region, and represent a significant public health threat (18, 39). Under natural conditions, most of the alphaviruses are transmitted by mosquitoes, in which they cause a persistent, life-long infection that has little effect on the biological functions of the vector. In vertebrates infected by mosquitoes during their blood meal, alphaviruses cause an acute infection, characterized by a viremia that is a prerequisite of infection of new mosquitoes and its circulation in nature.
Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the alphavirus genus. It continuously circulates in South, Central and North America and causes sporadic epidemics and epizootics that involve humans, horses and other domestic animals. During the most recent major outbreak in Venezuela and Colombia (1995) involving subtype IC VEEV, about 100,000 human cases occurred, with over 300 fatal encephalitis cases estimated (37). During VEEV epizootics, equine mortality due to encephalitis can reach 83%, and while the overall mortality rate is low in humans (<1%), neurological disease, including disorientation, ataxia, mental depression, and convulsions, can be detected in up to 14% of infected individuals, especially children (21). The human disease caused by VEEV is characterized as a febrile illness with chills, severe headache, myalgia, somnolence and pharyngitis. Young and old individuals develop a reticuloendothelial infection with severe lymphoid depletion, followed by encephalitis. The result of the CNS infection is an acute meningoencephalitis that leads to the death of neuronal cells (9). The neurologic signs appear within 4-10 days of the onset of illness and include seizures, paresis, behavioral changes and coma.
In spite of the continuous threat of VEEV epidemics, no safe and efficient vaccines have been designed for this virus. The attenuated TC-83 strain of VEEV was developed more than four decades ago by serial passage of a highly virulent Trinidad donkey (TRD) strain of VEEV in guinea pig heart cells (4). Presently, TC-83 is still the only available vaccine for laboratory workers and military personnel. Over 8,000 people have been vaccinated (2, 8, 34), and the cumulative data unambiguously demonstrate that nearly 40% of all vaccines develop a disease with some symptoms typical of natural VEE, including fever, systemic illness and other adverse effects (2). This TC-83 strain universally kills newborn, but not adult, mice after i.c. and s.c. inoculation (31), and is thus a good starting material for further attenuation and study of the effects of the mutations on viral pathogenesis.
The VEEV genome is a nearly 12-kb-long, single-stranded RNA molecule of positive polarity that mimics the structure of cellular mRNAs. The genome RNA contains both a 5′ methylguanylate cap and a 3′ polyadenylate tail (24), features which allow translation of viral proteins by host cell machinery immediately after release of the genome RNAs from the nucleocapsids. The 5′ two-thirds of the genome is translated into the nonstructural proteins (nsPs) that comprise the viral components of the replicative enzyme complex required for replication of the viral genome and transcription of the subgenomic RNA. The subgenomic RNA corresponds to the 3′ third of the genome. It is synthesized from the subgenomic promoter and translated into the viral structural proteins. The attenuated phenotype of the VEEV strain TC-83 is the result of two mutations in the strain TRD genome: one of them replaced an amino acid at position 120 in E2 glycoprotein, and the second changed nt 3 in the 5′UTR (11, 23, 24, 43). Thus, because of the alphavirus' very high mutation rate, the reversion of TC-83 to a pathogenic phenotype remains a great concern in the event that the appropriate selective conditions, such as virus passage in vivo, would occur. Moreover, VEEV TC-83 is capable of replicating in mosquito cells, and infecting mosquitoes following vaccination (32); therefore, its transmission by mosquitoes remain possible.
Ideally, live arbovirus vaccine strains should not be transmissible by arthropod vectors, because circulation among reservoir hosts could lead to unforeseen changes that might include increased virulence. This is especially true for attenuated strains, produced from wild-type viruses that rely on small numbers of attenuating mutations that may be subject to reversion, or for genetically modified strains that might evolve in unanticipated ways if they underwent vector-borne circulation. The former risk was underscored by the detection of the VEEV TC-83 vaccine strain in mosquitoes collected in Louisiana during 1971 (32), an area outside the epizootic/epidemic that was restricted to Texas.
The development of infectious cDNA for alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wt, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. Thus, such extensive mutations could prevent transmission of the genetically modified viruses by mosquito vectors.
Despite its importance as an emerging human and animal pathogen, its potential as a biological weapon and concerns about application of attenuated alphaviruses, the prior art is deficient in methods of generating attenuated strains of alphaviruses that are capable of replicating only in vertebrate cells. The present invention fulfills this long-standing need and desire in the art.